Stem cells have been the subject of intensive research due to potential for therapeutic applications such as tissue and organ regeneration. Pluripotent stem cells are capable of self renewal and differentiation into a diverse array of specialized cells. These can be further divided, by their derivation into adult and embryonic stem (ES) cells. ES cells are immortal cells obtained from the blastocyst inner call mass, which are able to differential into any cell of the three primary germ layers (pluripotent).
Adult stem cell populations can be found in the germline and somatic tissues, but are comparatively limited in abundance, self renewability and potency. While stem cells are a powerful tool in the examination of developmental and disease pathways, the great hope for ES cells has been invested primarily in regenerative therapy for genetic metabolic and degenerative disorders.
Somatic cell nuclear transfer (SCNT) has been used to insert the nuclei of specific somatic cells into enucleated oocytes to obtain ES cells that are nearly identical to the donor. While nuclear reprogramming has been achieved by fusion of ES with somatic cells (Yu, et al., 2006), the inefficiency of the fusion process is a major limiting factor. The major drawbacks of these methods are largely due to the embryonic derivation of the stem cells and the incidence of immune rejection.
To circumvent these issues, several groups have begun examining the trans-acting factors that enable reprogramming of somatic cell nuclei to a pluripotent state after SCNT or ES cell fusion. Taranger, et al., (2005) have been able to induce pluripotency in epithelial 293T cells by incubation with embryonic stem cell extracts. Takahashi and Yamanaka (2006) demonstrated that retroviral mediated transduction of four transcriptional factors, which are upregulated in ES cells, can reprogram murine adult and embryonic fibroblasts to a pluripotent state. The group screened several potential factors, finding that Oct3/4, Sox2, c-Myc and Klf4 were sufficient to induce pluripotent stem cells (iPS). The iPS cells showed gene expression and morphology similar to mES cells. Pluripotency of the fibroblast derived iPS cells was demonstrated by their ability to transmit through germ line (Okita, 2007).
Takahashi (2007) reprogrammed human dermal fibroblasts (HDF) to iPS using the same four factors. These HDF derived iPS also showed morphology, gene expression and teratoma formation similar to ES cells, as well as reduced methylation of oct4, rex1 and nanog promoters compared to the parental HDF cells. iPS-HDF were also able to undergo directed differentiation into neuronal cells, and even cardiomyocytes that began beating 12 days after induction of differentiation. Simultaneously, another group in Wisconsin reported that similar set of genes (oct4, sox2, lin28 and nanog) were sufficient to reprogram fetal lung fibroblasts and neonatal foreskin fibroblasts into iPS cells (Yu, 2007).
Unlike SCNT or ES-Somatic cell fusion methods of nuclear reprogramming, induction of pluripotency by introduction of defined transcription factors generates completely individual-specific stem cells, eliminating alteration of cell ploidy and the need for oocytes or previously existing stem cells. iPS cells can generate not only individual specific but also disease specific stem cells, allowing for enhanced testing of therapeutic drug efficacy.
Several laboratories have repeated these experiments, generating iPS from fibroblasts from mouse hepatocytes and gastric epithelial cell, and have demonstrated pluripotency by directed differentiation to several cell types, including neuronal, cardiovascular and hematopoietic lineages. Using a mouse model for sickle cell anemia, Hanna et al., (2007) generated iPS cells from autologous mouse skin cells, and repaired genetic mutations via homologous recombination. The repaired iPS cells were then directed to differentiation into hematopoietic progenitors, transplanted into the irradiated sickle cell anemia model mice and able to treat the disorder, illustrating the therapeutic potential of iPS cells.
While induction of pluripotency in somatic cells is at least a step forward in developing iPS cell for clinical application, significant concerns with the reported methods arise from the use of a retroviral vector. The integration site or number of copies of each gene transduced cannot be controlled. Uncontrolled genomic integration potentially interrupts critical genes, such as tumor suppressors, or alters transcriptional regulation of other genes, including oncogenes. Additionally, the use of c-myc, a known oncogene may contribute to the finding that 20% of the mice derived from iPS cells developed tumors (Okita, 2007). iPS cells have been successfully generated without the use of c-myc; however, the efficiency is significantly reduced (Nakagawa, 2008).
Okita (2008) and Stadtfeld (2008) utilized either adenoviral vector-mediated gene transfer or direct plasmid DNA transfection to generate iPS cells without an apparent trace of gene integration. However, as long as DNA molecules are introduced, the potential of DNA integration can not be totally eliminated. There remains a need for alternative methods for a safe clinical application of the iPS cells.
ES cells are pluripotent cells derived from the inner cell mass of the pre-implantation blastocyst. The distinguishing characteristics of ES cells, not possessed by other organ-specific stem cells, are the capacity to undergo robust self-renewal in cell culture while retaining a pluripotency for differentiation. These characteristics of ES cells have attracted attention for their use in cell-based transplantation therapy or tissue engineering; particularly after human ES cells were isolated in 1998.
To facilitate the use of pluripotent ES cells in cell transplantation, there are two major hurdles that need to be overcome. First, immune rejection by the recipients. Recently Induced pluripotent stem (iPS) cells, as discussed above, have yet to be developed as individual specific iPS cells. While establishing a human ES cell bank is also a valid alternative, a more difficult obstacle is the heterogenic nature of the cell population obtained from ES cell differentiation, primarily due to low efficiency of differentiation.
For most cell lineages, despite intensive studies in the field, it remains a difficult task to generate specific cell types from ES cells with high purity and efficiency. Before one can promote the development of cell types of interest from ES cells for clinical applications, there needs to be a consistent and reproducible record of successful lineage direction in vitro.
Three general approaches have been used to initiate ES cell differentiations. In the most popular method, ES cells are allowed to aggregate to form three-dimensional colonies known as embryoid bodies (EBs). Alternatively, ES cells are cultured directly on stromal cells and differentiation takes place upon contact with these cells. A third protocol involves differentiating ES cells in a monolayer on extracellular matrix proteins. Each of these three approaches has specific advantages and disadvantages, as reviewed by Keller et al. (2005).
Combining these three basic protocols, several differentiating protocols have been developed by varying serum concentration or by the use of a complex cocktail of growth factors and cytokines, extra cellular matrix proteins and small chemicals. Although these approaches have met with some success in neuroectodermal or neural specification (Ying, 2003, for example), it has proved difficult to enrich most of the other cell types, such as cardiomyocytes and hepatocytes, without the use of drug-resistant genes or fluorescent markers.
To differentiate ES cells for cell-based therapies, permanent genetic modification poses the danger of oncogenic transformation or other unforeseen changes upon implantation of the cells into an individual. The use of growth factors, cytokines and chemicals to drive ES cell differentiation into cardiomyocytes has had limited success, therefore a system was developed to directly introduce the regulatory proteins into ES cells to trigger the differentiation. Transfection reagent is toxic to the ES cells and the efficiency of transfection is low. And although protein factors can also be translocated into the host cells through a protein translocation domain (PTD), their translocation efficiency is generally low and limited to small sized proteins (Chauhan, 2007). This is further complicated by the fact that obtaining sufficient soluble protein is a challenge because most proteins tend to form inclusion bodies upon over expression.